Comprehensive RNA Polymerase II Interactomes Reveal Distinct and Varied Roles for Each Phospho-CTD Residue

Abstract

Summary Transcription controls splicing and other gene regulatory processes, yet mechanisms remain obscure due to our fragmented knowledge of the molecular connections between the dynamically phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD) and regulatory factors. By systematically isolating phosphorylation states of the CTD heptapeptide repeat (Y1S2P3T4S5P6S7), we identify hundreds of protein factors that are differentially enriched, revealing unappreciated connections between the Pol II CTD and co-transcriptional processes. These data uncover a role for threonine-4 in 3′ end processing through controlling the transition between cleavage and termination. Furthermore, serine-5 phosphorylation seeds spliceosomal assembly immediately downstream of 3′ splice sites through a direct interaction with spliceosomal subcomplex, U1. Strikingly, threonine-4 phosphorylation also impacts splicing through serving as a mark of co-transcriptional spliceosome release and ensuring efficient post-transcriptional splicing genome-wide. Thus, comprehensive Pol II interactomes identify the complex and functional connections between transcription machinery and other gene regulatory complexes.

Structure and dynamics of the RNAPII CTDsome with Rtt103

“…On average, each CTD heptad is phosphorylated once and the occurrence of two phosphorylations per repeat is a rare event (13,14). The coimmunoprecipitation of specific CTD phosphoisoforms revealed distinct functional sets of factors (CTDinteractome) related to each CTD phosphoisoform (15).…”

PHF13: A new player involved in RNA polymerase II transcriptional regulation and co-transcriptional splicing

“…S5P is required for efficient co-transcriptional splicing and has been reported to be involved in spliceosome assembly at the 5 0 and 3 0 splice sites triggering a splicing checkpoint and RNAPII pausing at intron-exon junctions. [15][16][17][29][30][31] Mammalian NET-seq demonstrated RNAPII S5P enrichment at 5 0 and 3 0 splice sites 15 and phospho-specific RNAPII immunoprecipitations have revealed that RNAPII S5P interacts with key proteins involved spliceosomal assembly. 15,16,29 For a detailed reviews on co-transcriptional splicing, we refer the reader to Saldi et al (2016) and Jonkers et al (2015) 25,32 .…”

“…[15][16][17][29][30][31] Mammalian NET-seq demonstrated RNAPII S5P enrichment at 5 0 and 3 0 splice sites 15 and phospho-specific RNAPII immunoprecipitations have revealed that RNAPII S5P interacts with key proteins involved spliceosomal assembly. 15,16,29 For a detailed reviews on co-transcriptional splicing, we refer the reader to Saldi et al (2016) and Jonkers et al (2015) 25,32 . Spliceosome assembly is a multi-step process that occurs on the pre-mRNA transcript, starting with recruitment of U1 snRNP at the 5 0 splice site 33 and U2 snRNP to the exonic nucleosome at the 3SS 34 and then at the branch point to form the pre-spliceosome (complex A).…”

“…4,8,[14][15][16] Thus, RNAPII S5P localizes to different chromatin regions, including repressed bivalent genes, 14 active promoters/promoter proximal regions 3 and at splice site junctions. 3,15 Furthermore, most of these RNAPII S5P contexts are affiliated with a pausing of RNAPII, 14,17,18 indicating that S5P is additionally important for co-transcriptional processing of pre-mRNA.…”

Extremely fast and incredibly close: cotranscriptional splicing in budding yeast

“…Recently, distinct next-generation sequencing approaches have measured the coupling of transcription to splicing in Saccharomyces cerevisiae: fast metabolic labeling with 4-thio-uracil (4tU-seq) (Barrass et al 2015), nascent RNAseq (Harlen et al 2016), and single molecule intron tracking (SMIT) (Carrillo Oesterreich et al 2016). These approaches produce, for many genes, quantitative estimates of the speed of splicing, extent of cotranscriptional splicing, or polymerase position at splicing, respectively.…”